Identification of Two Important Heme Site Residues (Cysteine 75 and Histidine 77) in CooA, the CO-Sensing Transcription Factor of Rhodospirillum rubrum

Abstract

The CO-sensing mechanism of the transcription factor CooA from Rhodospirillum rubrum was studied through a systematic mutational analysis of potential heme ligands. Previous electron paramagnetic resonance (EPR) spectroscopic studies on wild-type CooA suggested that oxidized (FeIII) CooA contains a low-spin heme with a thiolate ligand, presumably a cysteine, bound to its heme iron. In the present report, electronic absorption and EPR analysis of various substitutions at Cys residues establish that Cys75 is a heme ligand in FeIII CooA. However, characterization of heme stability and electronic properties of purified C75S CooA suggest that Cys75 is not a ligand in FeII CooA. Mutational analysis of all CooA His residues showed that His77 is critical for CO-stimulated transcription. On the basis of findings that H77Y CooA is perturbed in its FeII electronic properties and is unable to bind DNA in a site-specific manner in response to CO, His77 appears to be an axial ligand to FeII CooA. These results imply a ligand switch from Cys75 to His77 upon reduction of CooA. In addition, an interaction has been identified between Cys75 and His77 in FeIII CooA that may be involved in the CO-sensing mechanism. Finally, His77 is necessary for the proper conformational change of CooA upon CO binding.

A constitutively activated mutant of human soluble guanylyl cyclase (sGC): Implication for the mechanism of sGC activation

“…Appearance of this shoulder may reflect some transition to a mixed low-and high-spin coordinated heme, similar to thermal shift described for the mutant His-93-Cys myoglobin at Ϫ60°C (41). Alternatively, heme could form a new bond with a different proximal ligand (e.g., adjacent His-107 or Tyr-114), similar to the ligand switch suggested for the CooA transcription factor (42). Only additional studies will clarify this issue.…”

Dynamics of Carbon Monoxide Binding to CooA

“…Sample Preparation-CooA was purified as previously described (16), stored in 25 mM MOPS/0.1 M NaCl (pH 7.4), and then diluted into an appropriate buffer solution to give concentrations of ϳ2-5 M for the equilibrium binding titrations, ϳ5-25 M for the rapid mixing experiments, and ϳ50 M for time-resolved resonance Raman and microsecond laser photolysis experiments. CooA concentrations are expressed in terms of heme.…”

Mapping CooA·RNA Polymerase Interactions

“…Strains and Plasmids-The construction of E. coli strains expressing WT CooA and CooA variants and strains containing a chromosomal ␤-galactosidase reporter system was described previously (21). Sitedirected variants were constructed in a pKK223-3-based plasmid expression system as described previously (21).…”

“…Sitedirected variants were constructed in a pKK223-3-based plasmid expression system as described previously (21).…”

“…Previous studies have shown that CooA activates transcription at P cooF when expressed heterologously in vivo in Escherichia coli (21) and that CooA can interact with E. coli RNA polymerase at P cooF in vitro, dependent on CO and the ␣-CTD of RNAP (13). Furthermore, studies using RNAP variants with single substitutions in the ␣-CTD revealed that the contacts between CooA and the E. coli ␣-CTD are similar but not identical to those between CRP and ␣-CTD at class II promoters (13).…”